The Relationship Between SDF-1/CXCR4 Axis And Corneal Neovascularization Induced By Alkali Burn And Its Regulation Mechanism

Objective To investigate the expression of stromal cell-derived factor-1 (SDF-1) and its receptor,CXCR4, in rat cornea after alkali burn and its significance in corneal neovascularization. And to clarify the role of PKC- ζ in the regulation of SDF-1 stimulated VEGF mRNA transcription.Methods 1) Inflammatory corneal neovascularization model was established in Sprayue-Dawley rats with alkali burn. SDF-1/CXCR4 was detected in corneal samples on 2,5,7,10,14th day after cornea cautery by RT-PCR and Westernblot and immunohistochemistry method was used to explore the distribution of SDF-1/CXCR4 at rat cornea after alkali burn. 2) The model eyes were divided into two groups and each group contains 25 eyes. PBS and 15ug AMD3100 was subconjunctivally injected respectively in eyes immediately after burn once a day. The areas of corneal neovascularization were measured on 7,10,14th day after treatment and the expression of VEGF of two groups was investigated by westenblot. 3) Boyden chamber method was used to observe the chemotaxis of macrophage to SDF-1 and the effect of PKC inhibitor on this chemotaxis. The VEGF mRNA was detected by RT-PCR in SDF-1 stimulated U937 cell and the PKC regulation on this procedure. 4) 64 bp reverse repeated motifs of PKC- C target sequence were synthesized and inserted into the plasmid to construct the plasmid expressing shRNA- PKC- ζ and the plasmid was transfected into U937 cell line. RT-PCR was used to detect the expression of PKC- ζ. The basic and induced VEGF mRNA by SDF-1 was also examined by RT-PCR.Results 1) There are trace SDF-1 and CXCR4 in normal cornea on both mRNA and protein level. The expression of SDF-1 and CXCR4 increased on 2nd day and peaked on 7th day and began to decrease at 14th day after corneal cautery. The rat corneas were infiltrated by massive inflammatory cells adjacent to the cautery lesion. Inflammatory cells, corneal epithelial cells and keratocytes showed positive expression of SDF-1 and its receptor, CXCR4. 2) The average area of corneal neovascularization in therapeutic group on 7th day is 10.60 ± 2.31mm~2, significantly lower than 14.20 ± 3.42 mm~2 of control group. And on 10,14th day, the areas of treatment group were also significantly lower than those in control group. There was no significant reduction of VEGF expression between two groups. 3) SDF-1 could induce chemotaxis of macrophage in a dose-dependent manner and peaked at the concentration of 100ng/ml and VEGF mRNA transcription is up-regulated in SDF-1 stimulated U937 cell. Both of the two above-mentioned effects were effectively inhibited by a broad-spectrum PKC inhibitor, chelerythrine chloride, at different concentration and 10uM is the most effective. 4) The recombinant plasmid of shRNA-PKC- ζ was successfully constructed and the recombinant plasmid nearly completely suppress the PKC- ζ expression in U937 cell line. After transfection the basic VEGF and the SDF-1-induced VEGF both significantly reduced compared to control.Conclusions The interaction of SDF-1 derived from inflammatory cells, epithelial cells and corneal keratocytes and its receptor, CXCR4, participate in corneal wound repair and inflammatory corneal neovascularization. SDF-1 and VEGF synergistically induce and support the neovascularization in rat cautery cornea. AMD3100 could effectively inhibit the proliferation of inflammation-induced corneal neovascularization independent of VEGF. PKC is involved in the regulation of chemotaxis of macrophage induced by SDF-1 and especially PKC- ζ , may be involved in the regulation of VEGF transcription through NF-kappa B signal passageway in U937 cell line.